Vancomycin-intermediate Methods: Three pairs of VSSA/VISA clinical isolates have been isolated from persistent bloodstream infections during prolonged antibiotic therapy. Clinical pairs were compared for different features: i) biofilm formation ability using the crystal violet staining method (mature biofilm) and the Biofilm test based on measurement of superparamagnetic microbeads mobility trapped by biofilm (early biofilm), ii) cytotoxicity and immune response by quantifying lactate dehydrogenase (LDH) and Interleukin(IL)-6 release and iii) intracellular bacterial persistence using in vitro “lysostaphin protection” infection model of human osteoblasts.Aim
Method
Intracellular persistence of S. aureus is believed to be one of the major mechanisms leading to bone and joint infection (BJI) chronicity and relapses. Despite its poor intracellular activity, daptomycin (DAP) is increasingly used in the treatment of staphylococcal BJI. The well-known in vitro synergy of daptomycin with various betalactam antibiotics consequently led us to investigate whether these combinations enhance the activity of daptomycin against the intracellular reservoir of methicillin-susceptible (MSSA) and -resistant (MRSA) Osteoblastic MG63 cells were infected for 2h with MSSA strain or its isogenic MRSA. After killing the remaining extracellular bacteria with lysostaphin, infected cells were then incubated for 24h with DAP, oxacillin (OXA) or ceftaroline (CPT) alone or in combination, at the intraosseous concentrations reached with standard human therapeutic doses. Intracellular bacteria were then quantified by plating cell lysates. Minimum inhibitory concentrations (MICs) of these molecules alone and in combination were determined using the checkerboard method at pH7, but also at pH5 to mimic intracellular conditions.Aim
Method
Microbiological diagnosis of bone and joint infections (BJIs) currently relies on standard cultures which are time consuming and lack sensitivity. Various molecular approaches have been described and allowed improvement of BJI diagnosis. This study evaluated for the first time the performance of a DNA microarray-based assay (Prove-it™ Sepsis assay, PISA) for the rapid (<6 hours) detection and identification of 50 different species involved in BJI directly from clinical samples. We retrospectively selected 130 bone and joint samples (67 synovial fluids and 63 bone biopsies) including 114 positive and 16 negative samples. The microbiological diagnosis had been previously established either by culture(C+, n=53) or by PCR16S and sequencing when culture was negative (C-/PCR+). The positive samples were selected to match the species targeted on the DNA microarray. DNA extraction was performed before proceeding to PISA amplification and hybridization on every selected sample.Introduction
Material and methods
Rapid identification of bacteria from extemporaneous samples would greatly help management of prosthesis joint infection. The aim of the present retrospective study was to evaluate a new molecular assay (GeneXpert MRSA-SA SSTI (Cepheid)) for detecting Staphylococcus aureus (SA) and methicillin resistance directly from bone and joint samples in less an hour (58 minutes). Retrospective study using 91 frozen samples (76 patients) of joints (n=24), bone biopsies (n=42) and tissue biopsies (n=25): SA positive samples: n=72 (methicillin susceptible SA (MSSA), n=63; methicillin resistant MRSA, n=9) SA positive samples: n=19 The results were compared with routine results (culture in solid and liquid medium, identification and susceptibility test) from each participating lab.Introduction
Material et method
